Compounds that inhibit the interaction between signal-transducing proteins and the glgf (pdz/dhr) domain and uses thereof

ABSTRACT

This invention provides for a composition capable of inhibiting specific binding between a signal-transducing protein and a cytoplasmic protein. This invention also provides a method of identifying a compound capable of inhibiting specific binding between a signal-transducing protein and a cytoplasmic protein. This invention also provides a method of inhibiting the proliferation of cancer cells. This invention also provides a method of treating cancer with a composition in an amount effective to result in an amount in apoptosis of the cells. This invention also provides a method of inhibiting the proliferation of virally infected cells. This invention also provides for a method of treating a virally-infected subject with a composition in an amount effective to result in apoptosis of the cells. This invention also provides for pharmaceutical compositions.

[0001] The invention disclosed herein was made with Government supportunder Grant No. R01GM55147-01 from the National Institutes of Health ofthe United States Department of Health and Human Services. Accordingly,the U.S. Government has certain rights in this invention.

BACKGROUND

[0002] Throughout this application, various publications are referencedby author and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding Sequence Listing and the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

[0003] Fas (APO-1/CD95) and its ligand have been identified as importantsignal-mediators of apoptosis (Itoh, et al. 1991) The structuralorganization of Fas (APO-1/CD95) has suggested that it is a member ofthe tumor necrosis factor receptor superfamily, which also includes thep75 nerve growth factor receptor (NGFR) (Johnson, et al. 1986), theT-cell-activation marker CD27 (Camerini, et al. 1991), theHodgkin-lymphoma-associated antigen CD30 (Smith, et al. (1993), thehuman B cell antigen CD40 (Stamenkovic, et al. 1989), and T cell antigenOX40 (Mallett, et al. 1990). Genetic mutations of both Fas and itsligand have been associated with lymphoproliferative and autoimmunedisorders in mice (Watanabe-Fukunaga, et al. 1992; Takahashi, et al.1994).

[0004] Furthermore, alterations of Fas expression level have beenthought to lead to the induction of apoptosis in T-cells infected withhuman immunodeficiency virus (HIV) (Westendorp, et al. 1995).

[0005] Several Fas-interacting signal transducing molecules, such asFas-associated phosphatase-1 (FAP-1) (FIG. 1) (Sato, et al. 1995)FADD/MORT1/CAP-1/CAP-2 (Chinnaiyan, et al. 1995; Boldin, et al. 1995;Kischkel, et al. 1995) and RIP (Stanger, et al. 1995), have beenidentified using yeast two-hybrid and biochemical approaches. All butFAP-1 associate with the functional cell death domain of Fas andoverexpression of FADD/MORT1 or RIP induces apoptosis in cellstransfected with these proteins. In contrast, FAP-1 is the only proteinthat associates with the negative regulatory domain (C-terminal 15 aminoacids) (Ito, et al. 1993) of Fas and that inhibits Fas-inducedapoptosis.

[0006] FAP-1 (PTPN13) has several alternatively-spliced forms that areidentical to PTP-BAS/hPTP1E/PTPL1, (Maekawa, et al. 1994; Banville, etal. 1994; Saras, et al. 1994) and contains a membrane-binding regionsimilar to those found in the cytoskeleton-associated proteins, ezrin,(Gould et al. 1989) radixin (Funayama et al. 1991) moesin (Lankes, etal. 1991), neurofibromatosis type II gene product (NFII) (Rouleau, etal. 1993), and protein 4.1 (Conboy, et al. 1991), as well as in thePTPases PTPH1 (Yang, et al. 1991), PTP-MEG (Gu, et al. 1991), and PTPD1(Vogel, et al. 1993). FAP-1 intriguingly contains six GLGF (PDZ/DHR)repeats that are thought to mediate intra- and inter-molecularinteractions among protein domains. The third GLGF repeat of FAP-1 wasfirst identified as a domain showing the specific interaction with theC-terminus of Fas receptor (Sato, et al. 1995). This suggests that theGLGF domain may play an important role in targeting proteins to thesubmembranous cytoskeleton and/or in regulating biochemical activity.GLGF repeats have been previously found in guanylate kinases, as well asin the rat post-synaptic density protein (PSD-95) (Cho, et al. 1992),which is a homolog of the Drosophila tumor suppressor protein,lethal-(1)-disc-large-1 [dlg-1] (Woods, et al 1991; Kitamura, et al.1994). These repeats may mediate homo- and hetero-dimerization, whichcould potentially influence PTPase activity, binding to Fas, and/orinteractions of FAP-1 with other signal transduction proteins. Recently,it has also been reported that the different PDZ domains of proteinsinteract with the C-terminus of ion channels and other proteins (FIG. 1)(TABLE 1) (Kornau, et al. 1995; Kim, et al. 1995; Matsumine, et al.1996) TABLE 1 Proteins that interact with PDZ domains. C-terminalAssociated Protein sequence protein Reference Fas (APO-1/CD95) SLV FAP-12 NMDA receptor SDV PSD95 3 NR2 subunit Shaker-type K+ TDV PSD95 & DLG 4channel APC TEV DLG 5

SUMMARY OF THE INVENTION

[0007] This invention provides a composition capable of inhibitingspecific binding between a signal-transducing protein and a cytoplasmicprotein containing the amino acid sequence (G/S/A/E)—L—G—(F/I/L)(Sequence I.D. No.: 1). Further, the cytoplasmic protein may contain theamino acid sequence (K/R/Q)—X_(n)—(G/S/A/E)—L—G—(F/I/L) (Sequence I.D.No.: 2), wherein X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids and nrepresents at least 2, but not more than 4. In a preferred embodiment,the amino acid sequence is SLGI (Sequence I.D. No.: 3). Further, theinvention provides for a composition when the signal-transducing proteinhas at its carboxyl terminus the amino acid sequence (S/T)—X—(V/I/L)(Sequence I.D. No.: 4), wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, and the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids.

[0008] This invention also provides for a method of identifying acompound capable of inhibiting specific binding between asignal-transducing protein and a cytoplasmic protein containing theamino acid sequence (G/S/A/E)—L—G—(F/I/L). Further this inventionprovides for a method of identifying a compound capable of inhibitingspecific binding between a signal-transducing protein having at itscarboxyl terminus the amino acid sequence (S/T)—X—(V/L/I) and acytoplasmic protein.

[0009] This invention also provides for a method inhibiting theproliferation of cancer cells, specifically, where the cancer cells arederived from organs comprising the colon, liver, breast, ovary, testis,lung, stomach, spleen, kidney, prostate, uterus, skin, head, thymus andneck, or the cells are derived from either T-cells or B-cells.

[0010] This invention also provides for a method of treating cancer in asubject in an amount of the composition of effective to result inapoptosis of the cells, specifically, where the cancer cells are derivedfrom organs comprising the thymus, colon, liver, breast, ovary, testis,lung, stomach, spleen, kidney, prostate, uterus, skin, head and neck, orthe cells are derived from either T-cells or B-cells.

[0011] This invention also provides for a method of inhibiting theproliferation of virally infected cells, specifically wherein thevirally infected cells are infected with the Hepatitis B virus,Epstein-Barr virus, influenza virus, Papilloma virus, Adenovirus, HumanT-cell lymphtropic virus, type 1 or HIV.

[0012] This invention also provides a pharmaceutical compositioncomprising compositions capable of inhibiting specific binding between asignal-transducing protein and a cytoplasmic protein.

[0013] This invention also provides a pharmaceutical compositioncomprising compounds identified to be capable of inhibiting specificbinding between a signal-transducing protein and a cytoplasmic protein.

BRIEF DESCRIPTION OF THE FIGURES

[0014] As used herein, amino acid residues are abbreviated as follows:A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys;L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val;W, Trp; and Y, Tyr.

[0015]FIG. 1. Diagram of Fas-associated phosphatase-1 protein, showingthe six GLGF (PDZ/DHR) domain repeats; comparison of similar membranebinding sites with other proteins and proteins that contain GLGF(PDZ/DHR) repeats.

[0016]FIGS. 2A, 2B, 2C and 2D. Mapping of the minimal region of theC-terminal of Has required for the binding to FAP-1. Numbers at rightshow each independent clone (FIGS. 2C and 2D).

[0017]2A. Strategy for screening of a random peptide library by theyeast two-hybrid system.

[0018]2B. Alignment of the C-terminal 15 amino acids of Fas betweenhuman (Sequence I.D. No.: 5), rat (Sequence I.D. No.: 6), and mouse(Sequence I.D. No.: 7).

[0019]2C. The results of screening a semi-random peptide library. Toprow indicates the amino acids which were fixed based on the homologybetween human and rat. Dash lines show unchanged amino acids.

[0020]2D. The results of screening a random peptide library (SequenceI.D. No.: 8, Sequence I.D. No.: 9, Sequence I.D. No.: 10, Sequence I.D.No.: 11, Sequence I.D. No.: 12, Sequence I.D. No.: 13, Sequence I.D.No.: 14, Sequence I.D. No.: 15, Sequence I.D. No.: 16, Sequence I.D.No.: 17, respectively).

[0021]FIGS. 3A, 3B and 3C. Inhibition assay of Fas/FAP-1 binding vitro.

[0022]3A. Inhibition assay of Fas/FAP-1 binding using the C-terminal 15amino acids of Fas. GST-Fas fusion protein (191-355) was used for invitro binding assay (lane 1, 3-10). GST-Fas fusion protein (191-320)(lane 2) and 1 mM human PAMP (N-terminal 20 amino acids ofproadrenomedullin, M.W. 2460.9) (lane 3) were used as negative controls.The concentrations of the C-terminal 15 amino acids added were 1 (lane4), 3 (lane 5), 10 (lane 6), 30 (lane 7), 100 (lane 8), 300 (lane 9),and 1000 μM (lane 10).

[0023]3B. Inhibition assay of Fas/FAP-1 binding using the truncatedpeptides corresponding to the C-terminal 15 amino acids of Fas. Allsynthetic peptides were acetylated for this inhibition assay (SequenceI.D. No.: 4, Sequence I.D. No.: 18, Sequence I.D. No.: 19, Sequence I.D.No.: 20, Sequence ID. No.: 21, Sequence I.D. No.: 22, Sequence I.D. No.:23, respectively).

[0024]3C Inhibitory effect of Fas/FAP-1 binding using the scannedtripeptides.

[0025]FIGS. 4A, 4B, 4C and 4D.

[0026]4A. Interaction of the C-terminal 3 amino acids of Fas with FAP-1in yeast.

[0027]4B. Interaction of the C-terminal 3 amino acids of Fas with FAP-1in vitro.

[0028]4C. Immuno-precipitation of native Fas with GST-FAP-1.

[0029]4D. Inhibition of Fas/FAP-1 binding with Ac-SLV or Ac-SLY.

[0030]FIGS. 5A, 5B, 5C, 5D, 5E and 5F. Microinjection of Ac-SLV into theDLD-1 cell line. Triangles identify the cells both that were could bemicroinjected with Ac-SLV and that condensed chromatin identified. Onthe other hand, only one cell of the area appeared apoptotic whenmicroinjected with Ac-SLY.

[0031]5A. Representative examples of the cells microinjected with Ac-SLVin the presence of 500 ng/ml CH11 are shown in phase contrast.

[0032]5B. Representative examples of the cells microinjected with AC-SLYin the presence of 500 ng/ml CH11 are shown in phase contrast.

[0033]5C. Representative examples of the cells microinjected with Ac-SLVin the presence of 500 ng/ml CH11 are shown stained with FITC.

[0034]5D. Representative examples of the cells microinjected with AC-SLYin the presence of 500 ng/ml CH11 are shown stained with FITC.

[0035]5E. Representative examples of the cells microinjected with Ac-SLVin the presence of 500 ng/ml CH11 are shown with fluorescent DNAstaining with Hoechst 33342.

[0036]5F. Representative examples of the cells microinjected with AC-SLYin the presence of 500 ng/ml CH11 are shown in fluorescent DNA stainingwith Hoechst 33342.

[0037]FIG. 6. Quantitation of apoptosis in microinjected DLD-1 cells.

[0038]FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H.

[0039]7A. Amino acid sequence of human nerve growth factor receptor(Sequence I.D. No.: 24).

[0040]7B. Amino acid sequence of human CD4 receptor (Sequence I.D. No.25).

[0041]7C. The interaction of Fas-associated phosphatase-1 to theC-terminal of nerve growth factor receptor (NGFR) (p75).

[0042]7D. Amino acid sequence of human colorectal mutant cancer protein(Sequence I.D. No.: 26).

[0043]7E. Amino acid sequence of protein kinase C, alpha type.

[0044]7F. Amino acid sequence of serotonin 2A receptor (Sequence I.D.No.: 27).

[0045]7G. Amino acid sequence of serotonin 2B receptor (Sequence I.D.No.: 28).

[0046]7H. Amino acid sequence of adenomatosis polyposis coli protein(Sequence ID. No.: 29).

DETAILED DESCRIPTION OF THE INVENTION

[0047] As used herein, amino acid residues are abbreviated as follows:A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys;L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val;W, Trp; and Y, Tyr.

[0048] In order to facilitate an understanding of the material whichfollows, certain frequently occurring methods and/or terms are bestdescribed in Sambrook, et al., 1989.

[0049] The present invention provides for a composition capable ofinhibiting specific binding between a signal-transducing protein and acytoplasmic protein containing the amino acid sequence(G/S/A/E)—L—G—(F/I/L), wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,and each slash within such parentheses separating the alternative aminoacids. Further, the cytoplasmic protein may contain the amino acidsequence (K/R/Q)—X_(n)-(G/S/A/E)—L—G—(F/I/L), wherein X represents anyamino acid which is selected from the group comprising the twentynaturally occurring amino acids and n represents at least 2, but notmore than 4. Specifically, in a preferred embodiment, the cytoplasmicprotein contains the amino acid sequence SLGI.

[0050] The amino acid sequence (K/R/Q)—X_(n)—(G/S/A/E)—L—G—(F/I/L) isalso well-known in the art as “GLGF (PDZ/DHR) amino acid domain.” Asused herein, “GLGF (PDZ/DHR) amino acid domain” means the amino acidsequence (K/R/Q)—X_(n)-(G/S/A/E)—L—G—(F/I/L).

[0051] In a preferred embodiment, the signal-transducing protein has atits carboxyl terminus the amino acid sequence (S/T)—X—(V/I/L), whereineach — represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, and the X represents any aminoacid which is selected from the group comprising the twenty naturallyoccurring amino acids.

[0052] The compositions of the subject invention may be, but not limitedto, antibodies, inorganic compounds, organic compounds, peptides,peptidomimetic compounds, polypeptides or proteins, fragments orderivatives which share some or all properties, e.g. fusion proteins.The composition may be naturally occurring and obtained by purification,or may be non-naturally occurring and obtained by synthesis.

[0053] Specifically, the composition may be a peptide containing thesequence (S/T)—X—(V/I/L)—COOH, wherein each — represents a peptide bond,each parenthesis encloses amino acids which are alternatives to oneother, each slash within such parentheses separating the alternativeamino acids, the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids. Inpreferred embodiments, the peptide contains one of the followingsequences: DSENSNFRNEIQSLV, RNEIQSLV, NEIQSLV, EIQSLV, IQSLV, QSLV, SLV,IPPDSEDGNEEQSLV, DSEMYNFRSQLASVV, IDLASEFLFLSNSFL, PPTCSQANSGRISTL,SDSNMNMNELSEV, QNFRTYIVSFV, RETIESTV, RGFISSLV, TIQSVI, ESLV. A furtherpreferred embodiment would be an organic compound which has the sequenceAc—SLV—COOH, wherein the Ac represents an aceytl and each — represents apeptide bond.

[0054] An example of the subject invention is provided infra. Acetylatedpeptides may be automatically synthesized on an Advanced ChemTech ACT357using previously published procedures by analogy. Wang resin was usedfor each run and N^(α)-Fmoc protection was used for all amino acids, andthen 20% piperidine/DMF and coupling was completed using DIC/HOBt andsubsequently HBTU/DIEA. After the last amino acid was coupled, thegrowing peptide on the resin was acetylated with Ac₂O/DMF. Theacetylated peptide was purified by HPLC and characterized by FAB-MS and¹H-NMR.

[0055] Further, one skilled in the art would know how to constructderivatives of the above-described synthetic peptides coupled tonon-acetyl groups, such as amines.

[0056] This invention also provides for a composition capable ofinhibiting specific binding between a signal-transducing protein havingat its carboxyl terminus the amino acid sequence (S/T)—X—V/I/L), whereineach — represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, the X represents any amino acidwhich is selected from the group comprising the twenty naturallyoccurring amino acids, and a cytoplasmic protein.

[0057] The compositions of the subject invention includes antibodies,inorganic compounds, organic compounds, peptides, peptidomimeticcompounds, polypeptides or proteins, fragments or derivatives whichshare some or all properties, e.g. fusion proteins.

[0058] This invention also provides a method of identifying a compoundcapable of inhibiting specific binding between a signal-transducingprotein and a cytoplasmic protein containing the amino acid sequence(G/S/A/E)—L—G—(F/I/L), wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, which comprises (a) contacting the cytoplasmic protein bound tothe signal-transducing protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to displace the signal-transducing protein bound to thecytoplasmic protein and the bound cytoplasmic protein to form a complex;and (b) detecting the displaced signal-transducing protein or thecomplex formed in step (a) wherein the displacement indicates that thecompound is capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein.

[0059] The inhibition of the specific binding between thesignal-transducing protein and the cytoplasmic protein may affect thetranscription activity of a reporter gene.

[0060] Further, in step (b), the displaced cytoplasmic protein or thecomplex is detected by comparing the transcription activity of areporter gene before and after the contacting with the compound in step(a), where a change of the activity indicates that the specific bindingbetween the signal-transducing protein and the cytoplasmic protein isinhibited and the signal-transducing protein is displaced.

[0061] As used herein, the “transcription activity of a reporter gene”means that the expression level of the reporter gene will be alteredfrom the level observed when the signal-transducing protein and thecytoplasmic protein are bound. One can also identify the compound bydetecting other biological functions dependent on the binding betweenthe signal-transducing protein and the cytoplasmic protein. Examples ofreporter genes are numerous and well-known in the art, including, butnot limited to, histidine resistant genes, ampicillin resistant genes,β-galactosidase gene.

[0062] Further the cytoplasmic protein may be bound to a solid support.Also the compound may be bound to a solid support and comprises anantibody, an inorganic compound, an organic compound, a peptide, apeptidomimetic compound, a polypeptide or a protein.

[0063] An example of the method is provided infra. One can identify acompound capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein using directmethods of detection such as immuno-precipitation of the cytoplasmicprotein and the compound bound to a detectable marker. Further, onecould use indirect methods of detection that would detect the increaseor decrease in levels of gene expression. As discussed infra, one couldconstruct synthetic peptides fused to a LexA DNA binding domain. Theseconstructs would be transformed into the L40-strain with an appropriatecell line having an appropriate reporter gene. One could then detectwhether inhibition had occurred by detecting the levels of expression ofthe reporter gene. In order to detect the expression levels of thereporter gene, one skilled in the art could employ a variety ofwell-known methods, e.g. two-hybrid systems in yeast, mammals or othercells.

[0064] Further, the contacting of step (a) may be in vitro, in vivo, andspecifically in an appropriate cell, e.g. yeast cell or mammalian cell.Examples of mammalian cells include, but not limited to, the mousefibroblast cell NIH 3T3, CHO cells, HeLa cells, Ltk⁻ cells, Cos cells,etc.

[0065] Other suitable cells include, but are not limited to, prokaryoticor eukaryotic cells, e.g. bacterial cells (including gram positivecells), fungal cells, insect cells, and other animals cells.

[0066] Further, the signal-transducing protein may be a cell surfacereceptor, signal transducer protein, or a tumor suppressor protein.Specifically, the cell surface protein is the Fas receptor and may beexpressed in cells derived from organs including, but not limited to,thymus, liver, kidney, colon, ovary, breast, testis, spleen, lung,stomach, prostate, uterus, skin, head, and neck, or expressed in cellscomprising T-cells and B-cells. In a preferred embodiment, the T-cellsare Jurkat T-cells.

[0067] Further, the cell-surface receptor may be a CD4 receptor, p75receptor, serotonin 2A receptor, or serotonin 2B receptor.

[0068] Further, the signal transducer protein may be ProteinKinase-C-α-type.

[0069] Further, the tumor suppressor protein may be a adenomatosispolyposis coli tumor suppressor protein or colorectal mutant cancerprotein

[0070] Further, the cytoplasmic protein contains the amino acid sequenceSLGI, specifically Fas-associated phosphatase-1.

[0071] This invention also provides a method of identifying a compoundcapable of inhibiting specific binding between a signal-transducingprotein having at its carboxyl terminus the amino acid sequence(S/T)—X—(V/I/L), wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, the X represents any amino acid which is selected from the groupcomprising the twenty naturally occurring amino acids, and a cytoplasmicprotein which comprises (a) contacting the signal-transducing proteinbound to the cytoplasmic protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to displace the cytoplasmic protein hound to thesignal-transducing protein and bound signal-transducing protein to forma complex; and (b) detecting the displaced cytoplasmic protein or thecomplex of step (a), wherein the displacement indicates that thecompound is capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein. The inhibitionof the specific binding between the signal-transducing protein and thecytoplasmic protein affects the transcription activity of a reportergene. Further, in step (b), the displaced signal-transducing protein orthe complex is detected by comparing the transcription activity of areporter gene before and after the contacting with the compound in step(a), where a change of the activity indicates that the specific bindingbetween the signal-transducing protein and the cytoplasmic protein isinhibited and the cytoplasmic protein is displaced.

[0072] Further, in step (b), the displaced cytoplasmic protein or thecomplex is detected by comparing the transcription activity of areporter gene before and after the contacting with the compound in step(a), where a change of the activity indicates that the specific bindingbetween the signal-transducing protein and the cytoplasmic protein isinhibited and the signal-transducing protein is displaced.

[0073] As used herein, the “transcription activity of a reporter gene”means that the expression level of the reporter gene will be alteredfrom the level observed when the signal-transducing protein and thecytoplasmic protein are bound. One can also identify the compound bydetecting other biological functions dependent on the binding betweenthe signal-transducing protein and the cytoplasmic protein. Examples ofreporter genes are numerous and well-known in the art, including, butnot limited to, histidine resistant genes, ampicillin resistant genes,β-galactosidase gene.

[0074] Further, the cytoplasmic protein may be bound to a solid supportor the compound may be bound to a solid support, comprises an antibody,an inorganic compound, an organic compound, a peptide, a peptidomimeticcompound, a polypeptide or a protein.

[0075] An example of the method is provided infra. One could identify acompound capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein using directmethods of detection such as immuno-precipitation of the cytoplasmicprotein and the compound bound with a detectable marker. Further, onecould use indirect methods of detection that would detect the increaseor decrease in levels of gene expression. As discussed infra, one couldconstruct synthetic peptides fused to a LexA DNA binding domain. Theseconstructs would be transformed into L40-strain with an appropriate cellline having a reporter gene. One could then detect whether inhibitionhad occurred by detecting the levels of the reporter gene. Differentmethods are also well known in the art, such as employing a yeasttwo-hybrid system to detect the expression of a reporter gene.

[0076] Further the contacting of step (a) can be in vitro or in vivo,specifically in a yeast cell or a mammalian cell. Examples of mammaliancells include, but not limited to, the mouse fibroblast cell NIH 3T3,CHO cells, HeLa cells, Ltk⁻ cells, Cos cells, etc.

[0077] Other suitable cells include, but are not limited to, prokaryoticor eukaryotic cells, e.g. bacterial cells (including gram positivecells), fungal cells, insect cells, and other animals cells.

[0078] Further, the signal-transducing protein is a cell surfacereceptor, signal transducer protein, or a tumor suppressor protein.Specifically, the cell surface protein is the Fas receptor and isexpressed in cells derived from organs comprising thymus, liver, kidney,colon, ovary, breast, testis, spleen, stomach, prostate, uterus, skin,head and neck, or expressed in cells comprising T-cells and B-cells. Ina preferred embodiment, the T-cells are Jurkat T-cells.

[0079] Further, the cell-surface receptor may be a CD4 receptor, p75receptor, serotonin 2A receptor, or serotonin 2B receptor.

[0080] Further, the signal transducer protein may be ProteinKinase-C-α-type.

[0081] Further, the tumor suppressor protein may be a adenomatosispolyposis coli tumor suppressor protein or colorectal mutant cancerprotein.

[0082] Further, the cytoplasmic protein contains the amino acid sequenceSLGI, specifically Fas-associated phosphatase-1.

[0083] This invention also provides a method of inhibiting theproliferation of cancer cells comprising the above-describedcomposition, specifically, wherein the cancer cells are derived fromorgans including, but not limited to, thymus, liver, kidney, colon,ovary, breast, testis, spleen, stomach, prostate, uterus, skin, head andneck, or wherein the cancer cells are derived from cells comprisingT-cells and B-cells.

[0084] This invention also provides a method of inhibiting theproliferation of cancer cells comprising the compound identified by theabove-described method, wherein the cancer cells are derived from organsincluding, but not limited to, thymus, liver, kidney, colon, ovary,breast, testis, spleen, stomach, prostate, uterus, skin, head and neck,or wherein the cancer cells are derived from cells comprising T-cellsand B-cells.

[0085] The invention also provides a method of treating cancer in asubject which comprises introducing to the subject's cancerous cells anamount of the above-described composition effective to result inapoptosis of the cells, wherein the cancer cells are derived from organsincluding, but not limited to, thymus, liver, kidney, colon, ovary,breast, testis, spleen, stomach, prostate, uterus, skin, head and neck,or wherein the cancer cells are derived from cells comprising T-cellsand B-cells.

[0086] As used herein “apoptosis” means programmed cell death of thecell. The mechanisms and effects of programmed cell death differs fromcell lysis. Some observable effects of apoptosis are: DNA fragmentationand disintegration into small membrane-bound fragments called apoptoticbodies.

[0087] Means of detecting whether the composition has been effective toresult in apoptosis of the cells are well-known in the art. One means isby assessing the morphological change of chromatin using either phasecontrast or fluorescence microscopy.

[0088] The invention also provides for a method of inhibiting theproliferation of virally infected cells comprising the above-describedcomposition or the compound identified by the above-described, whereinthe virally infected cells comprise Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus, Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.

[0089] The invention also provides a method of treating avirally-infected subject which comprises introducing to the subject'svirally-infected cells the above-described composition effective toresult in apoptosis of the cells or the compound identified by theabove-described method of claim 27 effective to result in apoptosis ofthe cells, wherein the virally infected cells comprise the Hepatitis Bvirus, Epstein-Barr virus, influenza virus, Papilloma virus, Adenovirus, Human T-cell lymphtropic virus, type 1 or HIV.

[0090] Means of detecting whether the composition has been effective toresult in apoptosis of the cells are well-known in the art. One means isby assessing the morphological change of chromatin using either phasecontrast or fluorescence microscopy.

[0091] This invention also provides for a pharmaceutical compositioncomprising the above-described composition of in an effective amount anda pharmaceutically acceptable carrier.

[0092] This invention also provides for a pharmaceutical compositioncomprising the compound identified by the above-described method of inan effective amount and a pharmaceutically acceptable carrier.

[0093] This invention further provides a composition capable ofspecifically binding a signal-transducing protein having at its carboxylterminus the amino acid sequence (S/T)—X—(V/L/I), wherein each —represents a peptide bond, each parenthesis encloses amino acids whichare alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, and the X represents any aminoacid which is selected from the group comprising the twenty naturallyoccurring amino acids. The composition may contain the amino acidsequence (G/S/A/E)—L—G—(F/I/L), wherein each — represents a peptidebond, each parenthesis encloses amino acids which are alternatives toone other, and each slash within such parentheses separating thealternative amino acids. In a preferred embodiment, the compositioncontains the amino acid sequence (K/R/Q)—X_(n)—(G/S/A/E)—L—G—(F/I/L).wherein X represents any amino acid which is selected from the groupcomprising the twenty naturally occurring amino acids and n representsat least 2, but not more than 4. In another preferred embodiment, thecomposition contains the amino acid sequence SLGI.

[0094] This invention further provides a method for identifyingcompounds capable of binding to a signal-transducing protein having atits carboxyl terminus the amino acid sequence (S/T)—X—(V/L/I), whereineach — represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, the X represents any amino acidwhich is selected from the group comprising the twenty naturallyoccurring amino acids, which comprises (a) contacting thesignal-transducing protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to bind to the signal-transducing protein to form a complex;and (b) detecting the complex formed in step (a) so as to identify acompound capable of binding to the signal-transducing protein.Specifically, the identified compound contains the amino acid sequence(G/S/A/E)—L—G—(F/I/L). In a further preferred embodiment, the identifiedcompound contains the amino acid sequence SLGI.

[0095] Further, in the above-described method, the signal-transducingprotein may be bound to a solid support. Also, the compound may be boundto a solid support, and may comprise an antibody, an inorganic compound,an organic compound, a peptide, a peptidomimetic compound, a polypeptideor a protein.

[0096] Further, the signal-transducing protein may be a cell-surfacereceptor or a signal transducer. Specifically, the signal-transducingprotein may be the Fas receptor, CD4 receptor, p75 receptor, serotonin2A receptor, serotonin 2B receptor, or protein kinase-C-α-type.

[0097] This invention also provides a method of restoring negativeregulation of apoptosis in a cell comprising the above-describedcomposition or a compound identified by the above-described method.

[0098] As used herein restoring negative regulation of apoptosis meansenabling the cell from proceeding onto programmed cell death.

[0099] For example, cells that have functional Fas receptors andFas-associated phosphatase 1 do not proceed onto programmed cell deathor apoptosis due to the negative regulation of Fas by the phosphatase.However, if Fas-associated phosphatase 1 is unable to bind to thecarboxyl terminus of the Fas receptor ((S/T)—X—(V/L/I) region), e.g.mutation or deletion of at least one of the amino acids in the aminoacid sequence (G/S/A/E)—L—G—(F/I/L), the cell will proceed to apoptosis.By introducing a compound capable of binding to the carboxyl terminus ofthe Fas receptor, one could mimic the effects of a functionalphosphatase and thus restore the negative regulation of apoptosis.

[0100] This invention also provides a method of preventing apoptosis ina cell comprising the above-described composition or a compoundidentified by the above-described method.

[0101] This invention also provides a means of treating pathogenicconditions caused by apoptosis of relevant cells comprising theabove-described composition or the compound identified by theabove-described method.

[0102] This invention is illustrated in the Experimental Details sectionwhich follows. These sections are set forth to aid in an understandingof the invention but are not intended to, and should not be construedto, limit in any way the invention as set forth in the claims whichfollow thereafter.

EXPERIMENTAL DETAILS Example 1 Methods and Materials

[0103] 1. Screening a semi-random and random peptide library

[0104] To create numerous mutations in a restricted DNA sequence, PCRmutagenesis with degenerate oligonucleotides was employed according to aprotocol described elsewhere (Hill, et al. 1987). Based on the homologybetween human and rat, two palindromic sequences were designed forconstruction of semi-random library. The two primers used were5′-CGGAATTCNNNNNNNNNAACAGCNNNNNNNNNAATGAANNNCAAAGTCTGNN NTGAGGATCCTCA-3′(Seq. I.D. No.: 30) and5′-CGGAATTCGACTCAGAANNNNNNAACTTCAGANNNNNNATCNNNNNNNNNGT CTGAGGATCCTCA-3′(Seq. I.D. No.: 31). Briefly, the two primers (each 200 pmol), purifiedby HPLC, were annealed at 70° C. for 5 minutes and cooled at 23° C. for60 minutes. A Klenow fragment (5 U) was used for filling in with a dNTPmix (final concentration, 1 mM per each dNTP) at 23° C. for 60 minutes.The reaction was stopped with 1 μl of 0.5 M EDTA and the DNA waspurified with ethanol precipitation. The resulting double-stranded DNAwas digested with EcoRI and BamHI and re-purified by electrophoresis onnon-denaturing polyacrylamide gels. The double-strand oligonucleotideswere then ligated into the EcoRI-BamHI sites of the pBTM116 plasmid. Theligation mixtures were electroporated into the E. coli XL1-Blue MRF′(Stratagene) for the plasmid library. The large scale transformation wascarried out as previously reported. The plasmid library was transformedinto L40-strain cells (MATa, trp1, leu2, his3, ade2,LYS2:(lexAop)⁴-HIS3, URA3::(lexAop)⁶-lacZ) carrying the plasmid pVP16-31containing a FAP-1 cDNA (Sato, et al. 1995). Clones that formed onhistidine-deficient medium (His⁻) were transferred to plates containing40 μg/ml X-gal to test for a blue reaction product (β-gal⁻) in plate andfilter assays. The clones selected by His⁻ and β-gal⁻ assay were testedfor further analysis. The palindromic oligonucleotide,5′-CGGAATTC-(NNN)₄₋₁₅-TGAGGATCCTCA-3′ (Seq. I.D. No. 32), was used forthe construction of the random peptide library.

[0105] 2. Synthesis of peptides

[0106] Peptides were automatically synthesized on an Advanced ChemTechACT357 by analogy to published procedures (Schnorrenberg and Gerhardt,1989). Wang resin (C.2-0.3 mmole scale) was used for each run andN^(α)-Fmoc protection was employed for all amino acids. Deprotection wasachieved by treatment with 20% piperidine/DMF and coupling was completedusing DIC/HOBt and subsequent HBTU/DIEA. After the last amino acid wascoupled, the growing peptide on the resin was acetylated with Ac₂O/DMF.The peptide was cleaved from the resin with concomitant removal of allprotecting groups by treating with TFA. The acetylated peptide waspurified by HPLC and characterized by FAB-MS and ¹H-NMR.

[0107] 3. inhibition asssay of Fas/FAP-1 binding using the C-terminal 15amino acids of Fas.

[0108] HFAP-10 cDNA (Sato, et al. 1995) subcloned into the Bluescriptvector pSK-II (Stratagene) was in vitro-translated from an internalmethionine codon in the presence of ³⁵S—L-methionine using a coupled invitro transcription/translation system (Promega, TNT lysate) and T7 RNApolymerase. The resulting ³⁵S-labeled protein was incubated with GST-Fasfusion proteins that had been immobilized on GST-Sepharose 4B affinitybeads (Pharmacia) in a buffer containing 150 mM NaCl, 50 mM Tris [pH8.0], 5 mM DTT, 2 mM EDTA, 0.1% NP-40, 1 mM PMSF, 50 μg/ml leupeptin, 1mM Benzamidine, and 7 μg/ml pepstatin for 16 hours at 4° C. Afterwashing vigorously 4 times in the same buffer, associated proteins wererecovered with the glutathione-Sepharose beads by centrifugation, elutedinto boiling Laemmli buffer, and analyzed by SDS-PAGE and fluorography.

[0109] 4. Inhibition assay of terminal 15 amino acids of Fas andinhibitory effect of Fas/FAP-1 binding using diverse tripeptides.

[0110] In vitro-translated [³⁵S]HFAP-1 was purified with a NAP-5 column(Pharmacia) and incubated with 3 μM of GST-fusion proteins for 16 hoursat 4° C. After washing 4 times in the binding buffer, radioactivityincorporation was determined in a b counter. The percentage of bindinginhibition was calculated as follows: percent inhibition=[radioactivityincorporation using GST-Fas (191-335) with peptides−radioactivityincorporation using GST-Fas (191-320) with peptides]/[radioactivityincorporation using GST-Fas (191-335) without peptides−radioactivityincorporation using GST-Fas (191-320) without peptides]. n=3.

[0111] 5. Interaction of the C-terminal 3 amino acids of Fas with FAP-1in yeast and in vitro.

[0112] The bait plasmids, pBTM116 (LexA)-SLV, -PLV, -SLY, and -SLA, wereconstructed and transformed into L40-strain with pVP16-FAP-1 or -ras.Six independent clones from each transformants were picked up for theanalysis of growth on histidine-deficient medium. GST-Fas, -SLV, and PLVwere purified with GST-Sepharose 4B affinity beads (Pharmacia). Themethods for in vitro binding are described above.

[0113] 6. Immuno-precipitation of native Fas with GST-FAP-1 andinhibition of Fas/FAP-1 binding with Ac-SLV.

[0114] GST-fusion proteins with or without FAP-1 were incubated withcell extracts from Jurkat T-cells expressing Fas. The bound Fas wasdetected by Western analysis using anti-Fas monoclonal antibody (F22120,Transduction Laboratories). The tripeptides, Ac-SLV and Ac-SLY were usedfor the inhibition assay of Pas/FAP-1 binding.

[0115] 7. Microinjection of Ac-SLV into the DLD-1 cell line.

[0116] DLD-1 human colon cancer cells were cultured in RPMI 1640 mediumcontaining 10% FCS. For microinjection, cells were plated on CELLocate(Eppendorf) at 1×10⁵ cells/2 ml in a 35 mm plastic culture dish andgrown for 1 day. Just before microinjection, Fas monoclonal antibodiesCH11 (MBL International) was added at the concentration of 500 ng/ml.All microinjection experiments were performed using an automaticmicroinjection system (Eppendorf transjector 5246, micro-manipulator5171 and Femtotips) (Pantel, et al. 1995). Synthetic tripeptides weresuspended in 0.1% (w/v) FITC-Dextran (Sigma)/K-PBS at the concentrationof 100 mM. The samples were microinjected into the cytoplasmic region ofDLD-1 cells. Sixteen to 20 hours postinjection, the cells were washedwith PBS and stained with 10 μg/ml Hoechst 33342 in PBS. Afterincubation at 37° C. for 30 minutes, the cells were photographed and thecells showing condensed chromatin were counted as apoptotic.

[0117] 8. Quantitation of apoptosis in microinjected DLD-1 cells.

[0118] For each experiment, 25-100 cells were microinjected. Apoptosisof microinjected cells was determined by assessing morphological changesof chromatin using phase contrast and fluorescence microscopy (Wang, etal., 1995; McGahon, et al., 1995). The data are means +/− S.D. for twoor three independent determinations.

Discussion

[0119] In order to identify the minimal peptide stretch in theC-terminal region of the Fas receptor necessary for FAP-1 binding, an invitro inhibition assay of Fas/FAP-1 binding was used using a series ofsynthetic peptides as well as yeast two-hybrid system peptide libraries(FIG. 2A). First, semi-random libraries (based on the homology betweenhuman and rat Fas) (FIGS. 2B and 2C) of 15 amino acids fused to a LexADNA binding domain were constructed and co-transformed into yeast strainL40 with pVP16-31 (Sato, et al. 1995) that was originally isolated asFAP-1. After the selection of 200 His⁻ colonies from an initial screenof 5.0×10⁶ (Johnson, et al. 1986) transformants, 100 colonies that wereβ-galactosidase positive were picked for further analysis. Sequenceanalysis of the library plasmids encoding the C-terminal 15 amino acidsrevealed that all of the C-termini were either valine, leucine orisoleucine residues. Second, a random library of 4-15 amino acids fusedto a LexA DNA binding domain was constructed and screened according tothis strategy (FIG. 2D). Surprisingly, all of the third amino acidresidues from the C-termini were serine, and the results of C-terminalamino acid analyses were identical to the screening of the semi-randomcDNA libraries. No other significant amino acid sequences were found inthese library screenings, suggesting that the motifs of the last threeamino acids (tS—X—V/L/I) are very important for the association with thethird PDZ domain of FAP-1 and play a crucial role in protein-proteininteraction as well as for the regulation of Fas-induced apoptosis. Tofurther confirm whether the last three amino acids are necessary andsufficient for Fas/FAP-1 binding, plasmids of the LexA-SLV, -PLV, -PLY,-SLY, and -SLA fusion proteins were constructed and co-transformed intoyeast with pVP16-FAP-1. The results showed that only LexA-SLV associatedwith FAP-1, whereas LexA-PLV, -PLY, -SLY, and -SLA did not (FIG. 4A). Invitro binding studies using various GST-tripeptide fusions and invitro-translated FAP-1 were consistent with these results (FIG. 4B).

[0120] In addition to yeast two-hybrid approaches, in vitro inhibitionassay of Fas/FAP-1 binding was also used. First, a synthetic peptide ofthe C-terminal 15 amino acids was tested whether it could inhibit thebinding of Fas and FAP-1 in vitro (FIG. 3A). The binding of invitro-translated FAP-1 to GST-Fas was dramatically reduced and dependenton the concentration of the synthetic 15 amino acids of Fas. In contrastwith these results, human PAMP peptide (Kitamura, et al. 1994) as anegative control had no effect on Fas/FAP-1 binding activity under thesame biochemical conditions. Second, the effect of truncated C-terminalsynthetic peptides of Fas on Fas/FAP-1 binding in vitro was examined. Asshown in FIG. 3B, only the three C-terminal amino acids (Ac-SLV) weresufficient to obtain the same level of inhibitory effect on the bindingof FAP-1 to Fas as achieved with the 4-15 synthetic peptides.Furthermore, Fas/FAP-1 binding was extensively investigated using thescanned tripeptides to determine the critical amino acids residuesrequired for inhibition (FIG. 3C). The results revealed that the thirdamino acids residues from the C-terminus, and the C-terminal amino acidshaving the strongest inhibitory effect were either serine or threonine;and either valine, leucine, or isoleucine, respectively. However, therewere no differences among the second amino acid residues from theC-terminus with respect to their inhibitory effect on Fas/FAP-1 binding.These results were consistent with those of the yeast two-hybrid system(FIGS. 2C and 2D). Therefore, it was concluded that the C-terminal threeamino acids (SLV) are critical determinants of Fas binding to the thirdPDZ domain of FAP-1 protein.

[0121] To further substantiate that the PDZ domain interacts withtS/T—X—V/L/I under more native conditions, GST-fused FAP-1 proteins weretested for their ability to interact with Fas expressed in JurkatT-cells. The results revealed that the tripeptide Ac-SLV, but notAc-SLY, abolished in a dose-dependent manner the binding activity ofFAP-1 to Fas proteins extracted from Jurkat T-cells (FIGS. 4C and 4D).This suggests that the C-terminal amino acids tSLV are the minimumbinding site for FAP-1, and that the amino acids serine and valine arecritical for this physical association.

[0122] To next examine the hypothesis that the physiological associationbetween the C-terminal three amino acids of Fas and the third PDZ domainof FAP-1 is necessary for the in vivo function of FAP-1 as a negativeregulator of Fas-mediated signal transduction, a microinjectionexperiment was employed with synthetic tripeptides in a colon cancercell line, DLD-1, which expresses both Fas and FAP-1, and is resistantto Fas-induced apoptosis. The experiments involved the directmicroinjection of the synthetic tripeptides into the cytoplasmic regionsof single cells and the monitoring of the physiological response toFas-induced apoptosis in vivo. The results showed that microinjection ofAc-SLV into DLD-1 cells dramatically induced apoptosis in the presenceof Fas-monoclonal antibodies (CH11, 500 ng/ml) (FIGS. 5A, 5E and FIG.6), but that microinjection of Ac-SLY and PBS/K did not (FIGS. 5B, 5Fand FIG. 6). These results strongly support the hypothesis that thephysical association of FAP-1 with the C-terminus of Fas is essentialfor protecting cells from Fas-induced apoptosis.

[0123] In summary, it was found that the C-terminal SLV of Fas is alonenecessary and sufficient for binding to the third PDZ domain of FAP-1.Secondly, it is proposeed that the new consensus motif of tS/T—X—V/L/Ifor such binding to the PDZ domain, instead of tS/T—X—V. It is thereforepossible that FAP-1 plays important roles for the modulation of signaltransduction pathways in addition to its physical interaction with Fas.Thirdly, it is demonstrated that the targeted induction of Fas-mediatedapoptosis in colon cancer cells by direct microinjection of thetripeptide Ac-SLV. Further investigations including the identificationof a substrate(s) of FAP-1 and structure-function analysis will provideinsight to the potential therapeutic applications of Fas/FAP-1interaction in cancer as well as provide a better understanding of theinhibitory effect of FAP-1 on Fas-mediated signal transduction.

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What is claimed is:
 1. A composition capable of inhibiting specificbinding between a signal-transducing protein and a cytoplasmic proteincontaining the amino acid sequence (G/S/A/E)—L—F—(F/I/L), wherein each —represents a peptide bond, each parenthesis encloses amino acids whichare alternatives to one other, and each slash within such parenthesesseparating the alternative amino acids.
 2. The composition of claim 1,wherein the cytoplasmic protein contains the amino acid sequence(K/R/Q)—X_(n)—(G/S/A/E)—L—G—(F/I/L), wherein X represents any amino acidwhich is selected from the group comprising the twenty naturallyoccurring amino acids and n represents at least 2, but not more than 4.3. The composition of claim 1, wherein the cytoplasmic protein containsthe amino acid sequence SLGI.
 4. The composition of claim 1, wherein thesignal-transducing protein has at its carboxyl terminus the amino acidsequence (S/T)—X—(V/I/L), wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, and the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids.
 5. Thecomposition of claim 1, wherein the composition comprises an antibody,an inorganic compound, an organic compound, a peptide, a peptidomimeticcompound, a polypeptide, or a protein.
 6. The composition of claim 5,wherein the peptide comprises the sequence (S/T)—X—(V/I/L)—COOH, whereineach — represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, the X represents any amino acidwhich is selected from the group comprising the twenty naturallyoccurring amino acids.
 7. The composition of claim 6, wherein thepeptide has the amino acid sequence DSENSNFRNEIQSLV.
 8. The compositionof claim 6, wherein the peptide has the amino acid sequence RNEIQSLV. 9.The composition of claim 6, wherein the peptide has the amino acidsequence NEIQSLV.
 10. The composition of claim 6, wherein the peptidehas the amino acid sequence EIQSLV.
 11. The composition of claim 6,wherein the peptide has the amino acid sequence IQSLV.
 12. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceQSLV.
 13. The composition of claim 6, wherein the peptide has the aminoacid sequence SLV.
 14. The composition of claim 6, wherein the peptidehas the amino acid sequence IPPDSEDGNEEQSLV.
 15. The composition ofclaim 6, wherein the peptide has the amino acid sequenceDSEMYNFRSQLASVV.
 16. The composition of claim 6, wherein the peptide hasthe amino acid sequence IDLASEFLFLSNSFL.
 17. The composition of claim 6,wherein the peptide has the amino acid sequence PPTCSQANSGRISTL.
 18. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceSDSNMNMNELSEV.
 19. The composition of claim 6, wherein the peptide hasthe amino acid sequence QNFRTYIVSFV.
 20. The composition of claim 6,wherein the peptide has the amino acid sequence RETIESTV.
 21. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceRGFISSLV.
 22. The composition of claim 6, wherein the peptide has theamino acid sequence TIQSVI.
 23. The composition of claim 6, wherein thepeptide has the amino acid sequence ESLV.
 24. The composition of claim6, wherein the organic compound has the sequence Ac—SLV—COOH, whereinthe Ac represents an acetyl, each — represent a peptide bond.
 25. Acomposition capable of inhibiting specific binding between asignal-transducing protein having at its carboxyl terminus the aminoacid sequence (S/T)—X—(V/I/L), wherein each — represents a peptide bond,each parenthesis encloses amino acids which are alternatives to oneother, each slash within such parentheses separating the alternativeamino acids, the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids, and acytoplasmic protein.
 26. The composition of claim 25, wherein thecomposition comprises an antibody, an inorganic compound, an organiccompound, a peptide, a peptidomimetic compound, a polypeptide or aprotein.
 27. A method of identifying a compound capable of inhibitingspecific binding between a signal-transducing protein and a cytoplasmicprotein containing the amino acid sequence (G/S/A/E)—L—G—(F/I/L),wherein each — represents a peptide bond, each parenthesis enclosesamino acids which are alternatives to one other, each slash within suchparentheses separating the alternative amino acids, which comprises: (a)contacting the cytoplasmic protein bound to the signal-transducingprotein with a plurality of compounds under conditions permittingbinding between a known compound previously shown to be able to displacethe signal-transducing protein bound to the cytoplasmic protein and thebound cytoplasmic protein to form a complex; and (b) detecting thedisplaced signal-transducing protein or the complex formed in step (a),wherein the displacement indicates that the compound is capable ofinhibiting specific binding between the signal-transducing protein andthe cytoplasmic protein.
 28. The method of claim 27, wherein theinhibition of specific binding between the signal-transducing proteinand the cytoplasmic protein affects the transcription activity of areporter gene.
 29. The method of claim 28, where in step (b) thedisplaced signal-transducing protein or the complex is detected bycomparing the transcription activity of a reporter gene before and afterthe contacting with the compound in step (a), where a change of theactivity indicates that the specific binding between thesignal-transducing protein and the cytoplasmic protein is inhibited andthe signal-transducing protein is displaced.
 30. The method of claim 27,wherein the cytoplasmic protein is bound to a solid support.
 31. Themethod of claim 27, wherein the compound is bound to a solid support.32. The method of claim 27, wherein the compound comprises an antibody,an inorganic compound, an organic compound, a peptide, a peptidomimeticcompound, a polypeptide or a protein.
 33. The method of claim 27,wherein the contacting of step (a) is in vitro.
 34. The method of claim27, wherein the contacting of step (a) is in vivo.
 35. The method ofclaim 34, wherein the contacting of step (a) is in a yeast cell.
 36. Themethod of claim 34, wherein the contacting or step (a) is in a mammaliancell.
 37. The method of claim 27, wherein the signal-transducing proteinis a cell surface receptor.
 38. The method of claim 27, wherein thesignal-transducing protein is a signal transducer protein.
 39. Themethod of claim 27, wherein the signal-transducing protein is a tumorsuppressor protein.
 40. The method of claim 37, wherein the cell surfaceprotein is the Fas receptor.
 41. The method of claim 40, wherein the Fasreceptor is expressed in cells derived from organs comprising thethymus, liver, kidney, colon, ovary, breast, testis, spleen, stomach,prostate, uterus, skin, head and neck.
 42. The method of claim 40,wherein the Fas receptor is expressed in cells comprising T-cells andB-cells.
 43. The method of claim 37, wherein the cell-surface receptoris the CD4 receptor.
 44. The method of claim 37, wherein thecell-surface receptor is the p75 receptor.
 45. The method of claim 37,wherein the cell-surface receptor is the serotonin 2A receptor.
 46. Themethod of claim 37, wherein the cell-surface receptor is the serotonin2B receptor.
 47. The method of claim 38, wherein the signal transducerprotein is Protein Kinase-C-α-type.
 48. The method of claim 39, whereinthe tumor suppressor protein is adenomatosis polyposis coli tumorsuppressor protein.
 49. The method of claim 39, wherein the tumorsuppressor protein protein is the colorectal mutant cancer protein. 50.The method of claim 27, wherein the cytoplasmic protein contains theamino acid sequence SLGI, wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,and each slash within such parentheses separating the alternative aminoacids.
 51. The method of claim 40, wherein the cytoplasmic protein isFas-associated phosphatase-1.
 52. A method of identifying a compoundcapable of inhibiting specific binding between a signal-transducingprotein having at its carboxyl terminus the amino acid sequence(S/T)—X—(V/I/L), wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, the X represents any amino acid which is selected from the groupcomprising the twenty naturally occurring amino acids, and a cytoplasmicprotein, which comprises: (a) contacting the signal-transducing proteinbound to the cytoplasmic protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to displace the cytoplasmic protein bound to thesignal-transducing protein and the bound signal-transducing protein toform a complex; and (b) detecting the displaced cytoplasmic protein orthe complex of step (a) wherein the displacement indicates that thecompound is capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein.
 53. The methodof claim 52, wherein the inhibition of specific binding between thesignal-transducing protein and the cytoplasmic protein affects thetranscription activity of a reporter gene.
 54. The method of claim 53,where in step (b) the displaced cytoplasmic protein or the complex isdetected by comparing the transcription activity of a reporter genebefore and after the contacting with the compound in step (a), where achange of the activity indicates that the specific binding between thesignal-transducing protein and the cytoplasmic protein is inhibited andthe cytoplasmic protein is displaced.
 55. The method of claim 52,wherein the cytoplasmic protein is bound to a solid support.
 56. Themethod of claim 52, wherein the compound is bound to a solid support.57. The method of claim 52, wherein the compound comprises an antibody,an inorganic compound, an organic compound, a peptide, a peptidomimeticcompound, a polypeptide or a protein.
 58. The method of claim 52,wherein the contacting of step (a) is in vitro.
 59. The method of claim52, wherein the contacting of step (a) is in vivo.
 60. The method ofclaim 59, wherein the contacting of step (a) is in a yeast cell.
 61. Themethod of claim 59, wherein the contacting or step (a) is in a mammaliancell.
 62. The method of claim 52, wherein the signal-transducing proteinis a cell surface receptor.
 63. The method of claim 52, wherein thesignal-transducing protein is a signal transducer protein.
 64. Themethod of claim 52, wherein the signal-transducing protein is a tumorsuppressor protein.
 65. The method of claim 62, wherein the cell surfaceprotein is the Fas receptor.
 66. The method of claim 65, wherein the Fasreceptor is expressed in cells derived from organs comprising thethymus, liver, kidney, colon, ovary, breast, testis, spleen, stomach,prostate, uterus, skin, head and neck.
 67. The method of claim 65,wherein the Fas receptor is expressed in cells comprising T-cells andB-cells.
 68. The method of claim 62, wherein the cell-surface receptoris the CD4 receptor.
 69. The method of claim 62, wherein thecell-surface receptor is the p75 receptor.
 70. The method of claim 62,wherein the cell-surface receptor is the serotonin 2A receptor.
 71. Themethod of claim 62, wherein the cell-surface receptor is the serotonin2B receptor.
 72. The method of claim 63, wherein the signal transducerprotein is Protein Kinase-C-α-type.
 73. The method of claim 64, whereinthe tumor suppressor protein is adenomatosis polyposis coli tumorsuppressor protein.
 74. The method of claim 64, wherein the tumorsuppressor protein is the colorectal mutant cancer protein.
 75. Themethod of claim 52, wherein the cytoplasmic protein contains the aminoacid sequence SLGI, wherein each — represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,and each slash within such parentheses separating the alternative aminoacids.
 76. The method of claim 52, wherein the cytoplasmic protein isFas-associated phosphatase-1.
 77. A method inhibiting the proliferationof cancer cells comprising the composition of claim
 1. 78. The method ofclaim 77, wherein the cancer cells are derived from organs comprisingthe thymus, liver, kidney, colon, ovary, breast, testis, spleen,stomach, prostate, uterus, skin, head and neck.
 79. The method of claim77, wherein the cancer cells are derived from cells comprising T-cellsand B-cells.
 80. A method of inhibiting the proliferation of cancercells comprising the composition of claim
 25. 81. The method of claim80, wherein the cancer cells are derived from organs comprising thethymus, liver, kidney, colon, ovary, breast, testis, spleen, stomach,prostate, uterus, skin, head and neck.
 82. The method of claim 80,wherein the cancer cells are derived from cells comprising T-cells andB-cells.
 83. A method of inhibiting the proliferation of cancer cellscomprising the compound identified by the method of claim
 27. 84. Themethod of claim 83, wherein the cancer cells are derived from organscomprising the thymus, liver, kidney, colon, ovary, breast, testis,spleen, stomach, prostate, uterus, skin, head and neck.
 85. The methodof claim 83, wherein the cancer cells are derived from cells comprisingT-cells and B-cells.
 86. A method of inhibiting the proliferation ofcancer cells comprising the compound identified by the method of claim52.
 87. The method of claim 86, wherein the cancer cells are derivedfrom organs comprising the thymus, liver, kidney, colon, ovary, breast,testis, spleen, stomach, prostate, uterus, skin, head and neck.
 88. Themethod of claim 86, wherein the cancer cells are derived from cellscomprising T-cells and B-cells.
 89. A method of treating cancer in asubject which comprises introducing to the subject's cancerous cells anamount of the composition of claim 1 effective to result in apoptosis ofthe cells.
 90. The method of claim 89, wherein the cancer cells arederived from organs comprising the thymus, liver, kidney, colon, ovary,breast, testis, spleen, stomach, prostate, uterus, skin, head and neck.91. The method of claim 89, wherein the cancer cells are derived fromcells comprising T-cells and B-cells.
 92. A method of treating cancer ina subject which comprises introducing to the subject's cancerous cellsan amount of the composition of claim 25 effective to result inapoptosis of the cells.
 93. The method of claim 92, wherein the cancercells are derived from organs comprising the thymus, liver, kidney,colon, ovary, breast, testis, spleen, stomach, prostate, uterus, skin,head and neck.
 94. The method of claim 92, wherein the cancer cells arederived from cells comprising T-cells and B-cells.
 95. A method oftreating cancer in a subject which comprises introducing to thesubject's cancerous cells an amount of the compound identified by themethod of claim 27 effective to allow apoptosis of the cells.
 96. Themethod of claim 95, wherein the cancer cells are derived from organscomprising the thymus, liver, kidney, colon, ovary, breast, testis,spleen, stomach, prostate, uterus, skin, head and neck.
 97. The methodof claim 95, wherein the cancer cells are derived from cells comprisingT-cells and B-cells.
 98. A method of treating cancer in a subject whichcomprises introducing to the subject's cancerous cells an amount of thecompound identified by the method of claim 52 effective to result inapoptosis of the cells.
 99. The method of claim 98, wherein the cancercells are derived from organs comprising the thymus, liver, kidney,colon, ovary, breast, testis, spleen, stomach, prostate, uterus, skin,head and neck.
 100. The method of claim 98, wherein the cancer cells arederived from cells comprising T-cells and B-cells.
 101. A method ofinhibiting the proliferation of virally infected cells comprising thecomposition of claim
 1. 102. A method of inhibiting the proliferation ofvirally infected cells comprising the composition of claim
 25. 103. Amethod of inhibiting the proliferation of virally infected cellscomprising the compound identified by the method of claim
 27. 104. Amethod of inhibiting the proliferation of virally infected cellscomprising the compound identified by the method of claim
 52. 105. Themethod of claim 101, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 106. Themethod of claim 102, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 107. Themethod of claim 103, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 108. Themethod of claim 104, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 109. Amethod of treating a virally-infected subject which comprisesintroducing to the subject's virally-infected cells the composition ofclaim 1 effective to result in apoptosis of the cells.
 110. A method oftreating a virally-infected subject which comprises introducing to thesubject's virally infected cells the composition of claim 25 effectiveto result in apoptosis of the cells.
 111. A method of treating avirally-infected subject which comprises introducing to the subject'svirally-infected cells an amount of the compound identified by themethod of claim 27 effective to result in apoptosis of the cells.
 112. Amethod of treating a virally-infected subject which comprisesintroducing to the subject's virally-infected cells an amount of thecompound identified by the method of claim 52 effective to result inapoptosis of the cells.
 113. The method of claim 109, wherein thevirally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 114. The method of claim 110, whereinthe virally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 115. The method of claim 111, whereinthe virally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 116. The method of claim 112, whereinthe virally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 117. A pharmaceutical compositioncomprising the composition of claim 1 in an effective amount and apharmaceutically acceptable carrier.
 118. A pharmaceutical compositioncomprising the composition of claim 25 in an effective amount and apharmaceutically acceptable carrier.
 119. A pharmaceutical compositioncomprising the compound identified by the method of claim 27 in aneffective amount and a pharmaceutically acceptable carrier.
 120. Apharmaceutical composition comprising the compound identified by themethod of claim 52 in an effective amount and a pharmaceuticallyacceptable carrier.